DNA sequences to target proteins to the mammary gland for efficient secretion

ABSTRACT

Described is a method of targeting specific genes to the mammary gland which results in the efficient synthesis and secretion of biologically important molecules. Further, there is described as a composition of matter, a transgenic mammal having the ability to reproduce itself and being suitable for the secretion of biologically active agents into its milk. Additionally there is disclosed as a composition of matter, recombinant DNA gene complexes designed to integrate into a mammalian genome and to synthesize and secrete biological active agents into the milk. Furthermore methods of producing and using altered milk are disclosed.

This is a continuation of application Ser. No. 07/602,066, filed Oct.24, 1990, now U.S. Pat. No. 5,304,489, which is a continuation of Ser.No. 07/014,952, filed Feb. 17, 1987, now abandoned.

FIELD OF THE INVENTION

The present invention relates generally to transgenic mammals whichsecrete foreign compounds in their milk and to the method of producingtransgenic mammals with altered milk containing compounds useful in theareas of pharmacology, medicine, food and agricultural production andcancer research.

BACKGROUND OF THE INVENTION

Caseins are the principal milk proteins and are normally synthesized andsecreted only in the mammary gland during lactation. The first detailedcharacterization of the casein genes was done is the inventor'slaboratory. Yu-Lee, et al., Nuc. Acids Res., 14:1833-1902 (1986)

Since its introduction, microinjection of DNA into the pronucleus of afertilized one-cell embryo has been used to transfer a large number ofgenes into the mouse genome. Gordon et al. Proc. Natl. Acad. Sci. USA77:7380-7384 (1980); Palmiter and Brinster, Cell 41:343-345 (1985) andPalmiter and Brinster Ann. Rev. Genet. 20 465-499 (1986). The techniqueis useful for studies of the specific nucleotide sequences involved ingene expression and regulation, and for its practical applications forimprovement of domestic livestock. Transgenic sheep and pigs have nowbeen produced. Hammer et al., Nature (London) 315:343-345 (1985).Studies in cattle are in progress. Kraemer et al., In: Gene Transfer inCattle and Sheep, Banbury Report No. 20 pp. 221-227 (1985).

To produce transgenic animals of practical use in agriculture, theforeign gene must be integrated into the genome of the host animal andtransmitted to its offspring; it must be expressed in the appropriatetissue; and its expression must be at a high rate and subject to normalor artificial regulatory mechanisms. Tissue specificity of transgeneexpression has been reported for several genes including the ratelastase I gene, Ig light and heavy chain genes, the rat myosin lightchain gene and mouse/human β-globin gene; Swift et al., Cell 38:639-646(1984); Storb et al., Nature (London) 310:238-241 (1984), Grosscheldl etal., Cell 41:885-897 (1984); Shani, Nature (London) 314:283-286 (1985);and Chada, et al., Nature (London) 314:377-380 (1985). The factorsdirecting tissue-specific expression are not fully understood. Theevidence from the work with the MMTV promoter and the mousemetallothionein promoter suggests that DNA sequences in 5'-flanking DNAare important. Stewart et al., Nucl. Acids Res. 12:3895-3906 (1984) andPalmiter and Brinster, Cell 41:343-345 (1985).

Clues to this problem are beginning to emerge from studies both intransgenic animals and in cell culture systems. It is apparent thatspecific enhancer sequences in 5' flanking DNA, sometimes located farupstream from the transcription start site, and sequences in or close tothe promoter itself, are involved in tissue-specific gene expression.Gene expression in transgenic mice has been targeted to the appropriatetissue by inclusion of 5'-flanking and/or 3'-flanking DNA from thehomologous gene in the case of β-globin, elastase, α-fetoprotein,α-A-crystalline and insulin. Magram et al., Nature (London) 315:338-340(1985); Ornitz et al., Nature (London) 313:600-602 (1985); Krumlauf etal., Mol. Cell. Biol. 5:1639-1648 (1985); Overbeek et al., Proc. Natl.Acad. Sci. USA 82:7815-7819 (1985); and Hanahan, Nature (London)315:115-121 (1985).

The insulin gene has been analyzed the most extensively. The rat insulinI gene requires both an enhancer region between -103 and -133 and thepromoter region itself for expression of a marker gene in hamsterinsulinoma (HIT) cells compared to BHK cells. Edlund et al., Science230:912-916, (1985). Furthermore, the rat insulin II gene requires a 530bp 5'-flanking sequence to direct the expression of an SV40 oncogene tothe β cells of the pancreas in transgenic mice. Hanahan, Nature (London)315:115-121 (1985).

The bacterial chloramphenicol acetyltransferase (CAT) gene expressionhas been targeted to the eye lenses by linking a -364 to +45 DNAfragment of the murine α-A-crystalline to the coding sequence of the CATgene. Overbeek et al., Proc. Natl. Acad. Sci. USA 82; 7815-7819 (1985).

The ability to target specific genes to the mammary gland should resultin the efficient synthesis and secretion of proteins, ultimatelyimpacting the fields of biotechnology, pharmacology, medicine, foodscience and cancer research. For example, while a variety of expressionvectors have been developed for the efficient synthesis of proteins inbacteria and yeast, in many cases the biological activity of theseproteins is impaired because of the failure to correctly process theseproteins. Development of mammalian cell culture systems provides analternative strategy but the cost of these cell cultures may beprohibitive. The mammary gland provides a highly efficient in vivo modelfor the synthesis and secretion of grams of protein per day. Thesecretion continues during the lactation cycles of a mammals' life. Inaddition, the mammary gland contains the necessary post-translationalmodification systems required for the clevage, phosphorylation andglycosylation of proteins. Therefore, using this approach should make itpossible to efficiently synthesize and secrete biologically importantmolecules. For example, proteins, hormones, growth factors, drugs,lipids and carbohydrates can be synthesized and secreted, providing newtools in medicine and pharmacology. This methodology also provides amethod to manipulate the composition of mammary fluid (milk) by alteringits protein, carbohydrate and lipid composition and by the inclusion ofbacteriostatic agents. These changes will represent important changes inagricultural and food technology science. Additionally, the ability totarget oncogenes to the mammary gland will facilitate basic breastcancer research, because it provides a model to analyze the basicmechanisms of transformation in mammary epithelial cells. Thisinvestigational methodology is not available when using in vitro cellculture systems.

The present invention provides a method that not only targets theexpression of genes in the mammary gland but also provides forefficiently secreting these proteins during lactation.

SUMMARY OF THE INVENTION

An object of the present invention is a recombinant DNA gene complexwhich directs the synthesis of biological active agents into milk.

An additional object of the present invention is the development of atransgenic mammal which secretes biological active agents in its mammaryglands.

A further object of the present invention is the development of atransgenic mammal which secretes altered milk for use in pharmacology,medicine, cancer research, agriculture and food production.

Another object of the present invention is the development of atransgenic mammal which reproduces itself.

Thus, in accomplishing the foregoing objects there is provided inaccordance with one aspect of the present invention the provision as acomposition of matter, a recombinant DNA gene complex, comprising apromoter sequence, an enhancer sequence, a signal peptide sequence and acoding sequence derived from a gene coding for a biological activeagent. The promoter sequence, enhancer sequence and signal peptidesequence derive from at least one mammary gland-specific gene andfacilitate the expression of the coding sequence in the mammary gland.The coding sequence is selected from genes coding for biological activeagents.

A further aspect of the present invention is the development of theabove recombinant DNA gene complex, comprised further of a 5'untranslated mRNA sequence and a 3' untranslated mRNA sequence which areattached to the 5' and 3' ends respectively of the coding sequence. The5' and 3' flanking sequences increase the stability of the messenger RNAsynthesized by the recombinant DNA gene complex.

Another aspect of the present invention is the development as acomposition of matter a transgenic mammal for synthesizing peptides inthe mammary gland, comprising a germ line which includes a recombinantDNA gene complex; the germ line is transmittable to subsequentgenerations. Another aspect of the transgenic mammal is that it can beany mammal. The preferred embodiment is a non-human mammal.

A further aspect of the present invention is a method of targeting thesynthesis of peptides of at least one specific gene to the mammarygland, comprising the step of, inserting a recombinant DNA gene complexinto a germ line of a mammalian. Another embodiment includes a methodfor growing the embryo in an environment conducive to differentiationand development into a mammal. A further embodiment comprises the stepof confirming the stable incorporation of the gene complex into the germline. Another embodiment comprises the further step of testing themammary tissue and milk from the mammal for the expression of the codingsequence. An additional embodiment comprises the step of establishingthe proper functioning of the gene complex.

An additional aspect of the present invention is a method forconstructing a mammary gland specific gene complex, comprising the stepsof linking a promoter sequence, an enhancer sequence, and a signalpeptide sequence selected from mammary gland-specific genes, and acoding sequence from a gene which codes for a biological active agent.One embodiment the method comprises the further step of linking a 5'untranslated mRNA and a 3' untranslated mRNA sequence.

There is provided in accordance with another aspect of the presentinvention a method of synthesizing a biologically active agent inmammary gland comprising of steps of constructing a recombinant DNA genecomplex, inserting this gene complex into a germ line of a mammalianembryo, growing the embryo to maturity and testing milk produced by themammal containing the gene complex for the biological active material.

A further aspect of the present invention is a method of preventingspoilage in milk comprising the step of inserting a recombinant DNA genecomplex which includes a bacteriostatic coding sequence into the germline of a mammalian embryo.

Another aspect of the present invention is a method of examining themechanisms of mammary cancer comprising the steps of inserting arecombinant DNA gene complex which includes an oncogene into a germ lineof a mammalian embryo and mechanisticly analyzing the resultantdevelopment of cancerous tissues.

Another aspect of this invention is the development as a composition ofmatter a strain of transgenic mammals which secrete customized milk. Thecustomized milk can have altered concentrations of naturally occurringcompounds and/or can contain foreign compounds. The foreign compoundscan be drugs, hormones, peptides, proteins, lipids, carbohydrates andbacteriostatic agents. These foreign compounds are synthesized fromgenes derived from bacterial, animal and human genomes.

A further aspect of the present invention is a process for facilitatingthe production of dairy products comprising the step of incorporatingcustomized milk into the production of the dairy products.

An additional aspect of the present invention is a food productincluding customized milk produced from a transgenic mammal.

Further objects, features and advantages will be apparent from thefollowing description of preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. A recombinant DNA gene complex. E represents the enhancersequence; P represents the promoter sequence; signal peptide representsthe tissue specific sequence; cDNA represents the specific gene to besynthesized; the narrow line (-) represents flanking sequences; and thethick line () represents intronic sequences.

FIG. 1B. An alternative recombinant DNA gene complex. The symbols arethe same with the addition that 5'UT represents the 5' untranslated mRNAand 3'UT represents the 3' untranslated mRNA.

FIG. 2. Structure of the transferred rat β-casein gene. The diagramincludes the entire gene with about 1.3 Kb 5' flanking DNA and 1 Kb λDNA. This structure was isolated and subcloned from single phage clonesby a Kpn I-BAM HI digestion. Also shown is the 1.9 kb Eco RI probe usedto analyze the genomic DNA.

FIG. 3. Transfer of the Rat β-casein gene into transgenic mice. Analysisof the 1.9 Kb Kpn I-Bam HI fragment after insertion into mice embryos.The Rat and mouse DNA serve as controls.

FIG. 4. Limited pedigree of Rat β-casein gene in transgenic mouse 11.2.Circles represent females and squares represent males. The filled-insymbols indicate mice containing the rat β-casein gene. DNA blots oftail samples were performed on F₁ and F₂ generation mice.

FIG. 5. Expression of the β-casein gene in transgenic mice. Results ofRNA blots on RNA isolates from liver, brain, kidney. A specific S1nuclease protection assay using the 3' noncoding region of the ratβ-casein mRNA was used to distinguish rat and mouse mRNA's.

FIG. 6. Transfer of the Rat β-casein gene into transgenic mice. Agenomic clone containing the entire rat β-casein gene and 3.5 Kb of 5'and 3.0 Kb of 3' flanking DNA was inserted into mouse embryos. Five miceshow various numbers of copies of the transgene.

FIG. 7. RNase protection assay of mammary gland RNA from transgenicmice. RNA was extracted from lactating mammary tissue obtained as biopsysamples from three female F_(o) mice. Expression of the rat β-caseintransgene was detected in RNA using an RNase protection assay. Theletters represent as follows: Lane A (probe alone), lane B (0.5 μg ofrat lactating RNA), lane C (50 μg of lactating RNA from a noninjectedcontrol), Lanes D, E, and F (50 μg of RNA from positive transgenic mice)and lane G (50 μg of tRNA).

FIG. 8. Construction of casein-CAT fusion genes. The structure of thepSV_(o) CAT expression sector. Four β-casein-CAT and one γ-casein-CATfusion genes are shown containing up to 2.3 Kb of 5' flanking DNA and insome cases the 5' untranslated exon I and a portion of intron A of thesegenes. Numbering is relative to the casein mRNA CAP site designated as+1. The structural gene sequences are shown with exons in black andintrons in white.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

One embodiment of the invention as a composition of matter is arecombinant DNA gene complex including a promoter, enhancer, signalpeptide and coding sequences. In this combination the promoter, enhancerand signal peptide sequences are derived from mammary gland-specificgenes and the coding sequence codes for a biological active agent. Theusual method of constructing a mammary gland specific recombinant DNAgene complex includes the linking of a promoter, enhancer, signalpeptide and coding sequence together. FIG. 1A displays one embodiment ofthe invention showing the linking of a gene promoter sequence (P) withits enhancer sequence (E) by flanking sequences. These sequences arederived from genes which normally are specifically expressed only inmammary tissue. For example, these sequences can be obtained from geneswhich code for α-casein, β-casein, γ-casein, κ-casein, α-lactalbumin,β-lactoglobulin, and whey acidic protein.

The promoter-enhancer complex is then linked to a signal peptide exonsequence. A variety of signal peptide exons which are specific to themammary gland are available. The signal peptide exons play a role in theefficient translocation, recognition, removal and secretion of proteinsinto milk. Once the protein, carbohydrate, peptide or fat is secretedinto the milk, standard separation procedure can be used to purify thecomponents. Although the signal peptide will facilitatepost-translational processing, the intrinsic characteristics of somesynthesized molecules may prevent secretion into the milk. Thus, mammarytissue must be collected and the molecule of interest purified from thetissue. This approach is less satisfactory because the collection ofmammary tissue prevents the continual production of the compound ofinterest and the separation of components from tissue is a moredifficult procedure than separation from milk. Specific embodimentsemploy exons II of the α-, β-, and γ-casein genes and exon I of the wheyacidic protein gene.

The coding region (cDNA) of the gene of interest is attached to thepromoter-enhancer-signal peptide complex by an intron sequence. Thecoding region can be any gene or part a gene which codes for a molecule.It can include both intron and exon regions of the gene. For example,genes which code for proteins, milk proteins, lipids, carbohydrates,hormones, bacterial compounds (drugs, bacteriostatic agents),antibodies, antigens, and enzymes can be linked to thepromotor-enhancer-signal complex.

In the preferred embodiment the coding sequence is selected from genescoding for biological active agents selected from the group consistingof α-casein, β-casein, γ-casein, κ-casein, α-lactalbumin,β-lactoglobulin, whey acidic protein, hormones, drugs, proteins, lipids,carbohydrates, growth factors, chloramphenicol acetyltransferase andbacteriostatic agents. In a preferred embodiment the mammarygland-specific gene is selected from the group consisting of the genescoding for α-casein, β-casein, γ-casein, κ-casein, α-lactalbumin,β-lactoglobulin and whey acidic protein.

Another embodiment uses the same gene in deriving the promoter sequence,enhancer sequence and signal peptide sequence. Other specificembodiments employ the promoter, enhancer and signal peptide sequencesof the β-casein gene, and a coding sequence for either the β-casein geneor the chloramphenicol acetyltransferase gene.

An additional embodiment as a composition of matter is the recombinantDNA gene complex shown in FIG. 1B. This embodiment includes a 5'-untranslated sequence (5'UT) and a 3'-untranslated sequence (3' UT) of amessenger RNA (mRNA) linked to the coding region of the gene which isattached to the promotor-enhancer-signal complex. The untranslated mRNAsequences can be linked by introns. These untranslated mRNA sequencesare transcribed and are attached to the mRNA. These untranslated regionsaid in protecting the mRNA of the coding region from rapid breakdown.Naturally occurring genes whose mRNA shows a long half-life are goodcandidates for these untranslated regions. Examples of untranslatedregions which may be utilized in these constructions include theuntranslated mRNA sequences of the β-casein, β-globin and vitellogeninmRNA's. It has been found that the β-casein gene sequence provides apreferred embodiment.

The enhancer-promoter-signal peptide and enhancer-promoter-5'untranslated mRNA sequence--signal peptide 3' untranslated mRNA sequenceconstructs can be incorporated into a vector. Then the various cDNAs canbe incorporated whenever needed. The cDNAs are like cassettes beinginserted into a DNA sequence designed to specifically secrete compoundsinto milk. Thus a variety of recombinant DNA gene complexes can beeasily formed.

Once the recombinant DNA gene complex (foreign gene complex) is made,with or without untranslated sequences, it is integrated into the genome(germ line) of the host mammal. The integration of the foreign genecomplex into the germ line creates as a composition of matter, atransgenic animal. Furthermore, integration into the germ line allowsthe transmission of the foreign gene complex to offspring. Thus, astrain of mammals containing the foreign gene complex can be maintained.The foreign gene complex can be included into the genome of any mammal.In one preferred embodiment a non-human mammal is used.

The synthesis of biological active agents can be targeted to the mammarygland by inserting the recombinant DNA gene complex into the germ lineof a mammalian embryo. An additional embodiment includes the step ofinserting the embryo into an appropriate environment which is conduciveto differentiate and develop the embryo into a mammal. After the mammalis born the additional step of genome screening can be done to establishthat a stable incorporation of the foreign gene complex into the hostgenome has occurred. After the mammal reaches maturity, the lactatinggland can be examined to confirm that mRNA and/or molecule synthesis ofthe foreign gene complex is occurring in the mammary gland. This stepcan be used to establish the proper functioning of the recombinant genecomplex. Depending upon the characteristics of the foreign gene which isintegrated, a variety of screening procedures are available. Thescreening procedure can include probe analysis, mRNA analysis, enzymeanalysis, bacterial assays, antibody screens and protein, carbohydrateand lipid analysis.

In one preferred embodiment the foreign gone complex is inserted intothe germ line of a mammal at the one cell stage of an oocyte. If theintegration occurs at the one cell stage, a probe to the foreign genecomplex can be utilized to test any tissue, but, if integration occursat later stages in development the tissue to be examined is limited tothose developing from the cell line where integration occurs. Theinjected oocyte is then inserted into the oviducts of a host animal withthe same germ line.

Specific Examples of Foreign Gene Complexes

The 34.4 kb region of genomic DNA containing the 7.2 kb rat β-caseingene has been characterized. Jones et al J. Biol. Chem. 260:7042-7050(1985) the disclosure of which is incorporated by reference. The entiregene and either 1.3 or 2.3 kb of 5'-flanking DNA were isolated andsubcloned from single phage clones by either a Kpn I-Bam HI or BarnHI-Bam HI digestion. Jones et al. J Biol. Chem. 260:7042-7050 (1985).These constructions contain in addition 1 kb (KpmI-Bam HI) or 5 kb (BamHI-Bam HI) of λ DNA. FIG. 2 shows the KpnI-BamHI digestion fragmentcontaining the β-casein gene with 1 kb, 5' flanking DNA and 1 kb of λDNA. Alternatively, a 14.6 kb Bam HI-Bam HI fragment free of prokaryoticDNA can be isolated by ligation of a Bam HI-Sal I fragment from phageB12 to a Sal I-Bam HI fragment from phage B99. This constructioncontains 7kb of 5'-flanking DNA, the entire gene and 400 bp of3'-flanking sequence.

An example of a recombinant DNA gene complex includes a glucocorticoidresponse element (GRE) from mouse mammary tumor virus long terminalrepeat. This is inserted 5' to the mammary specific enhancer sequence(FIG. 1A). Its addition is facilitated by the addition of appropriaterestriction enzyme linkers. The GRE can be excised from plasmid pTK2A1by digestion with Xho II to generate a 340 bp fragment capable ofconferring glucocorticoid inducibility on the adjacent gene (Godowski etal., Nature 325: 365-368, (1987). The GRE permits an additional 10- to20-fold induction of the adjacent gene due to the elevatedglucocorticoid levels present during lactation.

One example of a recombinant DNA gene complex which can be used toelicit efficient tissue-specific expression in transgenic mice is theentire rat β-casein gene containing 7 kb of 5'-flanking DNA and lackingthe procaryotic vector sequences. The large and complex nature of thecasein genes, leaves few restriction enzyme sites available to excisethe λ DNA sequences without cleaving the gene at multiple sites. Thus,removal of the λ sequences from the Kpn I-Bam HI fragment requires Bal31 digestion followed by subcloning and DNA sequencing. Maniatis, etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Presspp. 207-209 (1982), the disclosure of which is incorporated byreference. Furthermore, the entire gene and its large flanking sequencescan be important for tissue-specific regulation.

An additional recombinant DNA gene complex was formed using β-casein-CATfusion genes. Bisbee and Rosen, ULCA Symposium on Molecular and CellularBiology "Transcriptional Control" (1986), the disclosure of which isincorporated by reference. This construction contains up to 2.3 kb of5'-flanking DNA. This can be conveniently excised from the vector DNAusing unique Nde I and Bam HI sites. The linear fragment free of vectorDNA is used. An alternative construction of the β-casein-CAT complex isthe ligation of additional 5' flanking sequences to the Xba I linkerused in the construction of the casein-CAT fusion genes. Only a few XbaI sites exist in the 7 kb of 5' flanking DNA therefore a Bam HI-Xba I(partial digestion) fragment is generated containing the missingupstream sequence. Another method of forming recombinant DNA genecomplexes when tissue-specific enhancer sequences are present within thegene is to screen for the enhancer sequences by assaying the restrictionendonuclease fragments spanning the gene and by cloning with appropriatelinkers into a vector. In the case of CAT the vector is a SV₁ CATvector. Gorman et al., Mol. Cell. Biol. 2:1044-1051 (1982), thedisclosure of which is incorporated by reference. This vector contains aconstitutive promoter from SV₄₀, but lacks the SV40 enhancer sequence.It is, therefore, useful for screening for promoter-independent enhanceractivity in different DNA fragments. Furthermore, a β-casein-CATconstruction containing only 511 bp of 5'-flanking DNA can be utilized.In the transgenic mice the unrearranged copies of the transferred genes,rat β-casein and CAT, are analyzed for expression.

In order to target expression to the mammary gland and to efficientlysecrete these proteins during lactation a signal peptide must be linkedto the complex. One example is the linkage of the 63 bp casein signalpeptide exon sequence in phase to CAT. The signal peptide for casein hasbeen shown to be highly conserved throughout mammalian evolution. Yu-Leeet al., 14:1883-1902 (1986), the disclosure of which is incorporated byreference. Although other signal peptides discussed above are availableit is advantageous to use a highly conserved sequence to facilitateefficient secretion in the mammary gland. Transfection of DNA encodingforeign secretory proteins into regulated secretory cells has shown thespecificity of protein sorting into secretory vesicles. Kelly, Science230:25-32 (1985) the disclosure of which is incorporated by reference.For example, a HindIII fragment containing the second exon (exon II) ofthe β-casein gene can be isolated. The HindIII sites are 14 bp 3' to the+2 amino acid of the mature casein and 548 bp 5' to the start of exonII, Jones et al., J. Biol. Chem. 260:7042-7050 (1985). To delete thetermination codon in phase with the ATG 3' to the +2 amino acid, Bal 31digestion is performed and HindIII linkers are inserted. This fragmentis inserted in the HindIII site of an SV₂ CAT vector Gorman et al., Mol.Cell. Biol. 2:1044-1051 (1982) and Rosen et al., In: Membrane Receptorsand Cellular Regulation Alan R. Liss, New York pp 385-396 (1985) thedisclosure of which is incorporated by reference.

An alternative approach is to synthesize directly a 45 bpoligonucleotide containing unique restriction enzyme linkers. This canbe ligated directly to the Hind III site in the β-casein-CAT vector. Theoligonucleotide approach is preferred since the cleavage sequences canbe controlled for better efficiency and precision.

The vector which is constructed or synthesized is transfected intoCOMMA-ID cells, and CAT is efficiently expressed. Bisbee and Rosen ULCASymposium on Molecular and Cellular Biology, "Transcriptional Control"(1986). This construction results in a CAT fusion protein containing anadditional 14 amino acids fused to the amino terminus of CAT followingcleavage of the signal peptide sequence.

In another preferred embodiment the previously determined cis-actingregulatory sequences required to elicit tissue-specific expression tothe casein signal peptide-CAT construction are linked to the aboveexample constructions. This is accomplished either by deleting the SV4072 bp enhancer from the above construction using Acc I and Sph I (thisgenerates essentially the SV₁ CAT vector) and inserting a mammaryspecific enhancer fragment, or by using partial HindIII digestion at anupstream HindIII site at -330 bp to generate a fragment containing boththe casein-specific promoter, exon I, intron A and exon II. In eithercase a linear DNA fragment lacking procaryotic vector sequences is usedto generate transgenic mice.

CAT activity can be determined by a variety of methods includingenzymatic analyses in the medium (milk), cytoplasmic and tissue extractsand by immunological assays employing a dot blot method.

The casein signal peptide sequence may not be sufficient to target thesecretion of all proteins, especially since secretion is dependent onmany factors for example intrinsic hydrophobicity. Thus, other signalpeptides or alterations to the flanking regions may be necessary toobtain secretion. The intrinsic signal peptide of a normally secretedprotein such as growth hormone or tissue plasminogen activator could beincluded instead of the casein signal peptide. Alternatively thecarboxy-terminal amino acids involved in anchoring the protein in themembrane may have to be deleted.

The authenticity of the example constructions can be confirmed by bothrestriction enzyme mapping and DNA sequencing.

Generation of Transgenic Mammals

Transgenic mammals can be generated by the process of incorporatingforeign DNA sequences into the host genome. This process consists ofembryo collection, injection of the DNA into the embryos, transfer ofthe surviving embryos to surrogate mothers, and screening the offspringfor integration and expression of the exogenous gene. Transgenic mammalscan include bacterial genes inserted into mammals to produce drugs,human genes inserted into non-human mammals to produce gram quantitiesof biological compounds in milk, human growth hormones incorporated intodairy animals, rat DNA incorporated into mouse, rat or bovine DNAincorporated into dairy animals, and DNA encoding goat sheep or pig milkproteins inserted into bovine.

Specific embodiments have included a method of preventing spoilage inmilk by the insertion of a recombinant DNA gene complex into a germ lineof a mammalian embryo. In the preferred embodiment the coding sequencefor a bacteriostatic agent is inserted. Additional embodiments includethe insertion of a recombinant DNA gene complex including an oncogeneinto the germ line of a mammalian embryo. The resultant transgenicmammal can be examined and the mechanism of the development of canceroustissues can be analyzed. This process also provided a procedure forfacilitating the production of dairy products by incorporatingcustomized milk into the production. The customized milk is produced ina transgenic mammal. The customized milk can include biological activeagents and can be used to produce a variety of products including food,drugs, cosmetics, hormones, carbohydrates, fats, amino acids andproteins.

Specific example of rat β-casein integration into mice.

1. Embryo collection

One cell embryos are collected by flushing the oviducts of female micewhich have been administered a superovulatory regime of gonadotropins.The gonadotropin regime is strain-dependent but essentially consists ofintraperitoneal (i.p.) injection of pregnant mare serum gonadotropin(PMS) followed by a later injection of human chronic gonadotropin (hCG)i.p. After the final gonadotropic administration, the female mouse ismated to a male mouse. The females are sacrificed approximately 18-20hrs after hCG administration the oviducts are flushed and the one cellembryos are made ready for injection.

In one specific embodiment approximately 14-18 g ICR females areinjected with approximately 5 I.U. PMS followed about 48 hours later byapproximately 5 I.U. injection hCG. Young immature mice respond betterto superovulation than older animals. Though either can be used. A maleB6 mousse is used in the mating.

2. Embryo injection

Embryos are placed in a drop of medium, Quinn, J. Reprod. Fert.66:161-168 (1982) the disclosure of which is incorporated by reference,and supplemented with 5 μg/ml cytochalasin B. The drop of medium iscovered with paraffin oil and the embryos are viewed with an invertedmicroscope using Hoffman optics. Injection of rat β-casein gene complexis accomplished by positioning the one cell embryo with a holdingmicropipette and injecting the rat β-casein gene complex into the malepronucleus with a finely pulled injection micropipette. The control ofthe fluid flow through the micropipettes consists of connectingStoelting micrometer syringes to the micropipettes with Teflon tubing.The entire system is filled with paraffin oil allowing positive pressurefor injection and negative pressure for holding the embryo to beinjected under fine control.

The rat β-casein gene complex to be injected is dissolved in a solutionof 10 mM Tris, pH 7.5, and 0.25 mM EDTA at a concentration of about 2ng/μl. Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442 (1985)the disclosure of which is incorporated by reference. Approximately 1-2pl of the β-casein gene complex solution is injected into thepronucleus. Embryo survival after injection is judged by the appearanceof normal morphology.

3. Embryo transfer

The embryos surviving microinjection are placed in HT6 medium inpreparation for transfer to the oviducts of 6- to 8-week old femalemice. The recipient is administered PMS i.p. followed later by hCG andplaced with a vasectomized male mouse. To aid the recipient in acceptingthe microinjection embryos the gonadotropic administration and matingcoincide with the schedule of the donor mouse.

In one example about 20-22g ICR females were injected with about 2 I.U.PMS j.p. followed about 48 hours later with 2 I.U. hCG i.p. Thosefemales with vaginal plugs after being placed with vasectomized maleswere used as recipients.

The recipient females are anesthetized, the oviducts are exposed withdorsolateral incision and the embryos are placed through the fimbrae ofthe oviduct with the use of a finely-pulled Pasteur pipette. Theoviducts are returned to the peritoneal cavity and the wound is closed.

The success of the embryo transfer is judged by the birth of mice about19-21 days after transfer. Success of the microinjection is assessed bySouthern hybridization analysis of DNA isolated from mouse tailbiopsies.

Specific example of bacterial CAT integration into mice.

1. Embryo collection - same procedures as under rat β-casein example.

2. Embryo injection

The same procedures as employed under the rat p-casein example are usedexcept that the bacterial CAT gene complex is injected into the malepronucleus of the one cell embryo. The bacterial CAT gene complex to beinjected is dissolved in a solution of 10 mM Tris, pH 7.5, and 0.25 mMEDTA as a concentration of about 2 ng/ul. Approximately 1-2 pl of thebacterial CAT gene complex solution is injected into the pronucleus.Embryo survival after injection is judged by the appearance of normalmorphology.

3. Embryo transfer

The same procedures employed for the β-casein gene complex are employed.The success of the embryo transfer is judged by the birth of mice about19-21 days after transfer. Success of the microinjection of thebacterial CAT is assessed by Southern hybridization analysis of DNAisolated from mouse tail biopsies.

Specific example recombinant DNA gene complex integration into cattle,sheep and pig embryos

The embryo collection and injection procedures are as previouslydescribed. Hammer et al. Nature (London) 315:343-345 (1985), and Kraemeret al In: Gene Transfer in Cattle and Sheep Banbury Report Nov. 20 pp.221-227 (1985) the. disclosure of which is incorporated by reference.The major difference between mice and cattle, sheep and pigs is in thevisualization of the pronuclei, in cattle, sheep and pigs the oocytesare not clear. Visualization is facilitated by centrifugation at about15,000 g for approximately 3 minutes. This centrifugation procedurestratifies the cytoplasm and leaves the pronuclei and nuclei visible byinterference contrast microscopy.

Analysis of Transferred Gene Structure and Expression

1. Isolation of DNA

A small tissue sample is homogenized with a Brinkman polytron in SETbuffer (150 mM NaCl, 20 mM Tris, pH 7.8, 1 mM Na₂ EDTA) at 37° C.overnight and extracted with phenol, phenol/chloroform/isoamyl alcohol,and chloroform/isoamyl alcohol. The DNA is recovered by ethanolprecipitation or spooling. The concentration of DNA is determined by thespecific fluorescence assay. Labarca and Paigen, Anal. Biochem.102:344-352 (1980) the disclosure of which is incorporated by reference.In mice about 1-2 cm of tail can be cut off and analyzed.

2. Southern and DNA dot blot assays

Initially the putative transgenic animal is screened for the presence ofthe transferred gene by Southern blotting. Ten μg of genomic DNA fromcontrol organisms supplying foreign DNA, control host and transfer hostare digested with restriction endonuclease, separated by agarose gelelectrophoresis, transferred to nitrocellulose, and hybridized with aunique gene probe.

For example when the rat β-casein gene is incorporated into mice a 1.9kb EcoRI gene probe is used. The status of the β-casein integration isanalyzed by digesting DNA with other restriction endonucleases, forexample, Kpn I and Bam HI, and probed with the 1.9 kb fragment as wellas with a 2.8 kb 5'-EcoRI fragment. The copy number of the transferredgene can be determined by a DNA dot blot assay using rat genomic DNAstandards (FIG. 3). Kafatos et al., Nucl. Acid Res. 7:1541-1553 (1979)the disclosure of which is incorporated by reference.

3. Mammary Gland Biopsy, RNA Isolation and Northern Blot

Lactating mammals are anesthetized and a biopsy of mammary gland tissueis removed and subjected to RNA extraction using the guanidinethiocyanate-CsCl method. Chirgwin et al., Biochemistry 18:5294-5299(1979) the disclosure of which is incorporated by reference. Mammarygland RNAs from biopsy samples and control tissues are separated byglyoxyai-agarose gel electrophoresis, transferred to nitrocellulose ornylon membranes and hybridized with a cRNA riboprobe. Zinn et al., Cell34:865-879 (1983) the disclosure of which is incorporated by reference.

For example, transgenic mice were anesthetized and the fourth mammarygland was removed and subjected to RNA extraction. The rat β-casein genemRNA was detected on nitrocellulose by hybridization with a rat 3'-cRNAriboprobe (FIG. 7).

4. RNase and S1 Nuclease Mapping

Correct initiation and termination of the transferred foreign genecomplex can be determined by RNase and S1 nuclease mapping,respectively.

For example, in rat β-casein gene RNase mapping, an 800 bp riboprobewhich covers the 5' flanking, the first exon, and a portion of intron A,is hybridized with RNA samples according to Zinn et al., Cell 34:865-879(1983) and subjected to RNase A and RNase T1 digestion. The protectedfragment is analyzed on an 8% polyacrylamide/urea sequencing gel.

For example, in rat β-casein gene S1 nuclease mapping, two differentprobes are used (FIG. 5). The first probe is Pvu II-Nco I fragmentlabeled at the 3' end by polynucleotide kinase. The second probe is anNco I-EcoRI genomic fragment which covers the 3' end of exon IX labeledat the 3' end by the Klenow fragment of DNA polymerase I. RNAs arehybridized with these probes, digested with S1 nuclease, and analyzed ona 5% polyacrylamide/urea gel. Maniatis, et al., Molecular Cloning: ALaboratory Manual, pp. 207-209 (1982). Each foreign gene which isincorporated will require its specific probe.

(e) CAT enzymetic and immunological assays

CAT enzymatic activity is assayed by the conversion of ¹⁴C-chloramphenicol to its acetylated derivatives. Gorman et al., Mol.Cell. Biol. 2:1044-1051 (1982). The results can be expressed as afunction of the DNA or protein content of the cells or tissue studied,and in some cases per copy number of the integrated CAT gene determinedby a DNA dot blot assay. CAT activity in milk is expressed per mg ofprotein. Alternatively CAT protein can be assayed using polyclonal ormonoclonal antibodies and a Western dot blot procedure. One skilled inthe art will recognize that other assays which detect either the proteinor its activity are available. The assay of CAT secretion in cellculture is performed using early passage COMMA-ID cells grown on afloating type I collagen gel in about 5% fetal bovine serum containinginsulin (approximately 5 μg/ml), hydrocortisone (approximately 1 μg/ml)and prolactin (approximately 1 μg/ml) for about 72 hrs followingdetachment of the gel. Under these conditions, β-casein mRNA representsapproximately 5-10% of the level observed in lactating tissue. Rosen etal., Annals N.Y. Acad. Sci. 478:63-76 (1986) the disclosure of which isincorporated by reference. Under comparable conditions casein isefficiently secreted from primary mouse mammary cells grown on afloating collagen gel. Lee et al., Proc. Natl. Acad. Sci USA82:1419-1423 (1985) the disclosure of which is incorporated byreference.

Analysis of Rat β-Casein incorporatin into mice

The principal difficulty with the analysis of the β-casein constructionsis the lack of a clonal cell line, which displays hormonally-regulatedcasein gene expression. Casein gene expression in primary cell culturesis dependent on cell-cell, as well as cell-substratum interactions.Levine and Stockdale J. Cell. Biol. 100:1415 (1985) and Lee et al.,Proc. Natl. Acad. Siv. USA 82:1419-1423 (1985). While both casein andwhey acidic protein (wap)gene expression can be regulated by hormones ina serum-free medium in explant cultures, WAP gene expression is notobserved in primary cultures or cell lines regardless of the cultureconditions employed. Hobbs et al., J. Biol. Chem. 257: 3598-3605. Thus,transgenic mice provide an alternative in vivo system in which toanalyze the functional role of cis-acting DNA sequences in the mammarygland.

Using the Kpn I-Bam HI fragment several transgenic mice have beengenerated (FIGS. 2 and 3). The transmission and expression of the ratβ-casein gene has been analyzed with a 1.9 kb Eco RI probe on genomicDNA blots. The specificity of the probe is shown by the observation thatonly weak cross-hybridization between the rat probe and a 10 kb mouseDNA Eco RI fragment is seen. Three different concentrations of ratgenomic DNA, a sample of mouse DNA and four DNAs isolated from differentF_(O) mice are shown in FIG. 3. One mouse 11.2 contained the expected1.9 kb fragment. More detailed analysis showed that approximately 4copies of the entire, unrearranged Kpn I-Bam HI fragment was present inmouse 11.2. The transmission of the integrated rat β-casein gene wasanalyzed by performing tail blots on a series of F₁ and F₂ mice assummarized in FIG. 4. Of the F₁ generation, 11 of 22 inherited the genewith an unaltered copy number suggesting a single site of integration.Two of the positive F₁ mice were bred in order to establish a homozygousline. Of the F₂ generation, 8 of 9 were positive and data suggests thatsome of these mice may be homozygous.

Mammary gland biopsies have been performed from lactating mice. Mouse11.2-2.4 was sacrificed and other tissues analyzed for casein geneexpression as well. Initially, RNA blots were performed using an SP6riboprobe synthesized from the 3' noncoding region of the rat β-caseinmRNA. Expression of the correctly-sized mRNA (1.1 kb) was observed onRNA blots in lactating RNA isolated from mice 11.2-2.0 and -2.4. Noexpression was detected in RNA isolated from liver and brain, and barelydetectable signal was seen in RNA isolated from kidney. Since the ratand mouse β-casein mRNAs are identical in size, a specific S1 nucleaseprotection assay was developed using the 3' noncoding region of the ratβ-casein mRNA. This probe was used to establish that the expression ofthe β-casein mRNA that was detected was due to the transferred rat geneand not the endogenous mouse gene.

A strand separated 448 NI probe was prepared, which had been labeled ata unique Nco I site. Protection from the mature mRNA yields a fragmentof 280 NT. If the pre-mRNA is not processed, a 144 NT fragment isgenerated. As shown in FIG. 5, 1 μg of RNA from lactating rat mammarygland gives a major band at 280 NT with a minor signal at 144 NT. Adiscrete signal of 280 NT is also seen in two of the transgenic mice(11.2-2.0 and -2.4), with a more intense band seen at 144 NT. Fifty μgof each RNA was assayed. No signals are seen in lactating RNA isolatedfrom either a control or negative transgenic mouse, or using tRNA in theassay. Upon longer exposure a faint signal of 280 NT was also observedin the RNA extracted from kidney, but not from liver. These results showthat the transferred rat β-casein gene was selectively expressed in thelactating mammary gland, but at a much lower level than the endogenousmouse gene. RNase and S1 protection experiments are used to determine ifthe rat β-casein gene transcripts are correctly initiated and processed.

Because of the reported inhibitory effects of prokaryotic vectorsequences on the level of tissue-specific transgene expression, and thepossibility of enhancer sequences located further 5' or 3' to the gene,a genomic clone, free of vector sequences, containing the entire ratβ-casein gene with 3.5 kb of 5' and 3.0 kb of 3' flanking DNA wasisolated and used to generate transgenic mice. As illustrated in FIG. 6,the expected 1.9 kb EcoRI DNA fragment is shown in five mice (3additional positive mice are not shown). RNA was extracted fromlactating mammary gland biopsies of three female F_(o) mice and caseingene expression analyzed as shown in FIG. 7 using a specific RNaseprotection assay. One of the three mice expressed the rat β-caseintransgene (FIG. 7, lane F). This mouse showed the expected 450 NTprotected fragment that was also observed in the control rat lactatingRNA sample (FIG. 7, lane B). Examination of F₁ generation has shown that7 of the 8 F_(o) transgenic females transmitted the foreign gene complexto their offspring.

These results demonstrate the transfer and expression of the ratβ-casein gene in transgenic mice. The levels of expression can beincreased by the addition of 5' and/or 3' sequences to elicit efficienttissue-specific gene expression. Even though the conservation of 5'flanking sequences was observed in the first few hundred bp upstream ofthe CAP site, this does not preclude other sequences outside of thisregion from having an important role in tissue-specific expression andregulation. While this is not the case in the majority of genes, asequence 5-7 kb upstream of the mouse α-fetoprotein gene has beenobserved to be necessary for efficient tissue-specific expression intransgenic mice. Hammer et al Science 235:53-58 (1987)

Analysis of CAT Incorporation

The pSV_(O) CAT expression vector contains the gene encoding thebacterial enzyme chloramphenicol acetyltransferase. Promotion orenhancement of gene expression by specific gene sequences can be readilyassayed by measuring CAT activity, a very sensitive enzymatic test thathas no background in eukaryotic cells. A series of β- and γ-casein-CATfusion genes have been constructed (FIG. 8). These have been assayed ina variety of mammary cell lines and primary cell cultures forhormonally-regulated promoter activity. Bisbee and Rosen, UCLA Symposiumon Molecular and Cellular Biology "Transcriptional Control" (1986). Theuse of casein-CAT fusion genes in transgenic animals provides a rapidand sensitive assay for tissue-specific promoter and enhancer function.

One skilled in the art will recognize other uses of the foreign genecomplex system for the secretion of proteins in the mammary gland andmilk. While presently preferred embodiments and examples of theinvention have been given for the purposes of disclosure, changestherein and other uses will occur to those skilled in the art which areencompassed within the spirit of the invention or defined by the scopeof the appended claims.

What is claimed is:
 1. A recombinant DNA complex to provide preferentialmammary gland expression and secretion of a selected polypeptidecomprising:(a) a casein promoter; (b) a transcribable DNA segment codingfor said selected polypeptide, wherein said promoter does not naturallycontrol the transcription of said DNA segment; (c) secretory DNAencoding a signal peptide functional in mammary secretory cells operablylinked between said promoter and said DNA segment to facilitatesecretion of said selected polypeptide; (d) a casein enhancer sequencecapable of conferring enhanced preferential transcription of saidrecombinant DNA complex in mammary secretory cells when said complex isintegrated into the genome of a non-human mammal, and (e) an intronicsequence from a casein gene or said transcribable DNA segment.
 2. Therecombinant DNA complex of claim 1 wherein said promoter is from acasein gene selected from the group consisting of α-casein, β-casein,γ-casein and κ-casein.
 3. The recombinant DNA complex of claim 2 whereinsaid promoter comprises the promoter from a β-casein gene.
 4. Therecombinant DNA complex of claim 3 wherein said casein enhancer isselected from the group consisting of α-casein, β-casein, γ-casein andκ-casein enhancers.
 5. The recombinant DNA complex of claim 1 furthercomprising a mouse mammary tumor virus glucocorticoid response element.6. The recombinant DNA complex of claim 1 wherein said secretory DNAencodes a signal peptide from a mammary gland specific gene.
 7. Therecombinant DNA complex of claim 6 wherein said mammary gland specificgene is selected from the group consisting of whey acid protein andcasein genes.
 8. The recombinant DNA complex of claim 7 wherein saidmammary gland specific gene is a casein gene.
 9. The recombinant DNAcomplex of claim 8 wherein said casein gene is selected from the groupconsisting of α-casein, β-casein, and κ-casein genes.
 10. Therecombinant DNA complex of claim 1 wherein said transcribable DNAsegment encodes a normally secreted polypeptide and said secretory DNAencodes the signal sequence of said secreted polypeptide.
 11. Therecombinant DNA complex of claim 1 further comprising:(f) DNA encoding a5' untranslated sequence from a mammary gland specific gene.
 12. Therecombinant DNA complex of claim 11 further comprising:(g) DNA encodinga 3' untranslated sequence.
 13. The recombinant DNA complex of claim 1,wherein said intronic sequence links said transcribable DNA segment tosaid promoter.
 14. The recombinant DNA complex of claim 1, wherein saidintronic sequence is located within said transcribable DNA segment. 15.The recombinant DNA complex of claim 13, wherein said intronic sequenceis a portion of an intron located between exons I and II of a ratβ-casein gene.
 16. A vector to provide preferential mammary glandexpression and secretion of a selected polypeptide comprising:(a) acasein promoter; (b) a transcribable DNA segment coding for saidselected polypeptide, wherein said promoter does not naturally controlthe transcription of said DNA segment; and (c) secretory DNA encoding asignal peptide functional in mammary secretory cells operably linkedbetween said promoter and said DNA segment to facilitate secretion ofsaid selected polypeptide, (d) a casein enhancer sequence capable ofconferring enhanced preferential transcription of said recombinant DNAcomplex in mammary secretory cells when said complex is integrated intothe genome of a non-human mammal, and (e) an intron from a casein gene.17. A recombinant DNA complex to provide preferential mammary glandexpression and secretion of a selected polypeptide comprising:(a) acasein promoter; (b) a transcribable DNA segment coding for saidselected polypeptide, wherein said promoter does not naturally controlthe transcription of said DNA segment; and (c) secretory DNA encoding asignal peptide functional in mammary secretory cells operably linkedbetween said promoter and said DNA segment to facilitate secretion ofsaid selected polypeptide, (d) a casein enhancer sequence capable ofconferring enhanced preferential transcription of said recombinant DNAcomplex in mammary secretory cells when said complex is integrated intothe genome of a non-human mammal, (e) an intron from a casein gene; and(f) a restriction enzyme linker sequence.
 18. A recombinant DNA complexto provide preferential mammary gland expression and secretion of aselected polypeptide comprising:(a) a casein promoter; (b) atranscribable DNA segment coding for said selected polypeptide, whereinsaid promoter does not naturally control the transcription of said DNAsegment; and (c) secretory DNA encoding a signal peptide functional inmammary secretory cells operably linked between said promoter and saidDNA segment to facilitate secretion of said selected polypeptide, (d) acasein enhancer sequence capable of conferring enhanced preferentialtranscription of said recombinant DNA complex in mammary secretory cellswhen said complex is integrated into the genome of a non-human mammal,and (e) an intron from a casein gene; wherein the recombinant DNAcomplex is linear.